The 5' cap structure of eukaryotic mRNA is crucial for translation and degradation resistance. Fluorophosphate-containing cap structures regulate mRNA turnover by modifying the 5'-5' oligophosphate bridge, which has been confirmed by literature to enhance nuclease resistance and serve as an essential tool for studying RNA metabolism.
Creative Biolabs' Custom Fluorophosphate-Containing Cap Service improves mRNA stability and protein expression through advanced chemical modification of the 5' oligophosphate bridge, addressing issues such as rapid in vivo degradation and low translation efficiency. It provides mRNA constructs that withstand harsh biological environments, helping to improve preclinical and clinical outcomes.
The Fluorophosphate-Containing Cap Service represents the zenith of mRNA 5' end engineering, specifically targeting the limitations of natural and early-generation synthetic caps.
Fig.1 The structures at the 5' end of RNA include capeless (5'-triphosphate) RNA, monomethylguanosine (m7G) mRNA caps, triomethylguanosine (m3G) caps, and γ-O-methyl caps found in snRNA, and "cap-like" structures found in bacterial RNA, etc.1
The core structure involves incorporating a fluorophosphate moiety into the triphosphate (ppp) bridge linking the m7G to the first transcribed nucleotide. This modification subtly alters the chemical environment of the phosphate linkages. Mechanistically, this change is critical because it disrupts the active site recognition or catalysis of RNA phosphohydrolases. Specifically, the fluorophosphate group hinders the enzyme-substrate complex formation for the human mRNA decapping scavenger (DcpS), which otherwise rapidly hydrolyzes the cap, exposing the mRNA to 5' exonucleases. This targeted disruption leads to superior chemical stability and a substantial increase in transcript half-life.
To ensure a high-quality, tailored solution, our process is meticulously structured, transparent, and designed for efficient integration into your drug development pipeline.
| Step | Activity Involved |
|---|---|
| Project Scoping & Input | Review of the client's current mRNA sequence, IVT conditions, and target application (vaccine, gene therapy). Selection of specific fluorophosphate analogs. |
| Cap Analog Synthesis & QC | Chemical synthesis of the chosen fluorophosphate cap analog. Rigorous Quality Control (QC) via HPLC and NMR to confirm purity and structure. |
| Optimized IVT and Capping | Execution of In Vitro Transcription (IVT), incorporating the fluorophosphate cap via co-transcriptional or post-transcriptional enzymatic labeling. |
| Purification & Polishing | Multi-step purification (e.g., RP-HPLC) to remove residual unincorporated nucleotides, enzymes, and double-stranded RNA (dsRNA) byproducts. |
| Comprehensive QC & Delivery | Final analysis, including capping efficiency check, integrity check (agarose gel, Bioanalyzer), concentration (UV spectroscopy), and endotoxin testing. |
Required Starting Materials: To start the custom service, clients usually provide: 1) Full target mRNA coding sequence (ORF) and UTR sequences; 2) Desired poly(A) tail length; 3) IVT plasmid DNA template.
Final Deliverables: After completion, you will receive: Chemically modified mRNA construct, Comprehensive Analytical QC Report (details capping efficiency and purity), and Synthesis Protocol Document (outlines key process parameters).
Estimated Timeframe: Typically, 4-8 weeks, depending on mRNA sequence complexity and synthesis scale. Optimizing a novel fluorophosphate analog may prolong this period.
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Custom Cap Analog Synthesis
We provide end-to-end 5' cap engineering, offering custom synthesis of fluorophosphate and phosphorothioate cap analogs precisely tailored to your mRNA sequence and therapeutic requirements (e.g., specific LNP compatibility).
Guaranteed DcpS Resistance
Delivery of mRNA constructs proven to provide superior protection against the DcpS decapping scavenger enzyme, a major limiter of in vivo half-life, ensuring prolonged protein expression.
Integrated Analytical Labeling
Option to incorporate spectroscopic or fluorescent tags directly into the phosphate bridge for unparalleled real-time analytical tracking, mechanistic study of cap-binding, and mRNA localization.
Highest Purity Standards
RP-HPLC purification protocols guaranteeing minimal dsRNA and truncated species, resulting in a final product purity suitable for IND-enabling studies and highly reduced risk of innate immune activation.
Comprehensive Capping Strategies
Expert consultation and implementation of advanced Cap 1 and Cap 2 modifications (both co- and post-transcriptional) to maximize translational efficiency alongside phosphate bridge stabilization.
Scalability and QC Documentation
Seamless transition from research quantities to large-scale cGMP-ready synthesis, backed by detailed, transparent Quality-by-Design (QbD) documentation.
A: The improvement is highly dependent on your specific sequence and delivery system (LNP), but published data indicate that phosphate-modified caps can significantly increase resistance to nucleases and decapping complexes like DcpS. We typically observe a marked extension of half-life in vivo, crucial for low-dose therapeutics. We recommend discussing your specific stability requirements with our chemists during a consultation.
A: Absolutely. Our cap modification protocols are fully compatible with the incorporation of modified nucleotides like Pseudouridine (Ψ). We view mRNA optimization holistically; combining enhanced cap stability with reduced immunogenicity from Ψ incorporation is our recommended strategy for achieving the highest protein expression and clinical safety profile.
A: This is a critical question. While complex, our proprietary methods and rigorous RP-HPLC purification protocols are specifically designed to minimize impurities. In fact, our process often results in lower dsRNA content and superior capping efficiency (approaching 100%) compared to standard commercial kits, ensuring your final product is translationally active and minimally immunogenic.
Moving from mRNA sequence to effective therapy needs more than standard IVT—it requires advanced chemical engineering. Creative Biolabs' Fluorophosphate-Containing Cap Service delivers key stability, analytical capability, and translational efficiency to overcome conventional mRNA limitations. With integrated construct design, advanced capping (Fluorophosphate, Phosphorothioate) and LNP formulation expertise, we're your one-stop partner to maximize next-gen mRNA therapeutic potential.
Contact Our Team for More Information to Discuss Your Project| Cat. No | Product Name | Promoter |
|---|---|---|
| CAT#: GTVCR-WQ001MR | IVTScrip™ pT7-mRNA-EGFP Vector | T7 |
| CAT#: GTVCR-WQ002MR | IVTScrip™ pT7-VEE-mRNA-EGFP Vector | T7 |
| CAT#: GTVCR-WQ003MR | IVTScrip™ pT7-VEE-mRNA-FLuc Vector | T7 |
| CAT#: GTVCR-WQ87MR | IVTScrip™ pT7-VEE-mRNA-Anti-SELP, 42-89-glycoprotein Vector | T7 |
| Cat. No | Product Name | Type |
|---|---|---|
| CAT#: GTTS-WQ001MR) | IVTScrip™ mRNA-EGFP (Cap 1, 30 nt-poly(A)) | Reporter Gene |
| CAT#: GTTS-WK18036MR | IVTScrip™ mRNA-Human AIMP2, (Cap 1, Pseudo-UTP, 120 nt-poly(A)) | Enzyme mRNA |
| (CAT#: GTTS-WQ004MR) | IVTScrip™ mRNA-Fluc (Cap 1, 30 nt-poly(A)) | Reporter Gene |
| (CAT#: GTTS-WQ009MR) | IVTScrip™ mRNA-β gal (Cap 1, 30 nt-poly(A)) | Reporter Gene |
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